It is widely recognized that in the United States almost 8 million people per year have burns or suffer from chronic, non-healing wounds (Singer & Clark (1999) N. Engl. J. Med. 341(10):738-746). Chronic wounds invariably occur in individuals with underlying disease and impaired wound healing is a hallmark of diabetic complications. Chronic wounds generally fall into three principal categories: venous stasis ulcers, diabetic ulcers and pressure ulcers. While the prevalence of these conditions vary, it is estimated that as many as 12 million people are afflicted with all forms of chronic wounds in the principal industrialized markets.
Approximately 800,000 diabetic foot ulcers are treated in the United States each year, of which 30% do not respond to standard care. Given that 15-20% of patients with foot ulcers will require amputation, these data underscore the medical benefit and cost saving that may be achieved through emerging technologies and therapies addressing chronic wound repair as provided by this invention.
Wound care and healing technology is evolving rapidly with new product offerings that respond to medical needs in both the acute and chronic wound management setting. According to the most recent National Health Interview Survey (NHIS), the incidence of burn injury causing wound stands at 4.2/10,000 or about 1.2 million annually. While severe wounds attributable to burns are significant, a staggering population of greater than 6 million people is afflicted with chronic ulcerations resulting from dermatological conditions and chronic ulcerations secondary to diabetes, immunosuppression and immobilization. Both acute and chronic wounds represent a major health problem, and a new generation of novel agents for wound healing are based on topical growth factors and protease inhibition therapy (Nwomeh, et al. (1998) Clin. Plast. Surg. 25:341-56).
Wound healing is dynamic process. The wound environment is variable depending on the health status of the individual and the underlying pathology. Knowledge of the physiology of the normal wound healing cycle through the phases of hemostasis, inflammation, granulation and maturation provides a framework for developing novel single or combination therapies.
In animal models of the disease, certain recombinant growth factors only partially enhance wound repair associated with diabetic state, however this efficacy may be increased using growth factor combinations such as TGF beta and basic FGF or TGF beta and PDGF. Consistent with this finding is the observation that combination of certain growth factors in fibroblasts cultured from human diabetic ulcers reproducibly cause a greater mitogenic response compared to any agent alone (Loot, et al. (2002) Eur. J. Cell Biol. 81(3):153-160). Moreover, topical applications of IGFs promote wound healing, in particular in association with IGF-binding proteins (Galiano, et al. (1996) J. Clin. Invest. 98(11):2462-8). Poor chronic wound healing in diabetic rats is associated with increased proteolytic enzymes, reduced IGF levels and destruction of IGF-binding proteins (Cechowska-Pasko, et al. (1996) Acta Biochimica Polonica 43(3):557-65). It is generally accepted that intervening parameters such as prolific proteolytic activity in the wound are deleterious to proper healing by degrading de novo granulation tissue and local growth factors and cytokines. Common proteases in the wound microenvironment are neutrophil elastase, matrix metalloproteases and plasmin (Cullen, et al. (2002) Wound Repair Regen. 10(1):16-25).
In burn-wound fluids, elastase from neutrophils is responsible for fibronectin degradation (Grinnell & Zhu (1994) J. Invest. Dermatol. 103(2):155-61). Neutrophil elastase activity is ten- to forty-fold higher in fluids from chronic wounds as compared with fluids from acute wounds. API, also known as alpha-1-antitrypsin, the major physiological inhibitor of neutrophil elastase, is conspicuously absent and not functional in chronic wounds (Rao, et al. (1995) J. Invest. Dermatol. 105(4):572-8). Moreover, in a case report of a patient with complete API deficiency, major skin abnormalities were observed (Ledoux-Corbusier & Achten (1975) J. Cutan. Pathol 2(1):25-9), compatible with a phenotype of uncontrolled elastase activity. Furthermore, in a pilot study, six patients with atopic dermatitis, who had failed to respond to high potency topical steroids, showed a remarkable improvement in tissue healing after topical application of API (Wachter & Lezdey (1992) Ann. Allergy 69(5):407-14).
Site-directed mutagenesis of the anti-elastase API active site suggests that the growth-promoting activity of API may be governed by a sequence that is distinct from its enzyme inhibitory domain (Sandoval, et al. (2002) Protein Eng. 15:413-8). The endogenous 36 amino acid C-terminal fragment of API (C36) is produced after proteolytic cleavage of the inhibitor. This is the only section of API (independent of the active site inhibitory loop) known to have biological activity. This peptide, which has been isolated from spleen and bile (Johansson, et al. (1992) FEBS Lett. 299(2):146-8) and shown to have chemotactic activity (Stockley, et al. (1990) Am. J. Respir. Cell Mol. Biol. 2(2):163-70).